Working with embryonic stem cells and trophoblast stem cells, we investigated the role of geminin in preventing DNA re-replication during cell proliferation and differentiation at the beginning of mouse development. The dual roles of geminin during trophoblast proliferation and differentiation. Geminin is a protein involved in both DNA replication and cell fate acquisition. Although it is essential for mammalian preimplantation development, its role remains unclear. We showed that ablation of the Geminin gene in trophoblast stem cells (TSCs) proliferating in the presence of FGF4 closely mimics the events triggered by FGF4 deprivation: arrest of cell proliferation, formation of giant cells, excessive DNA replication in the absence of DNA damage and apoptosis, and changes in gene expression that include loss of Chk1 with up-regulation of p57 and p21. Moreover, FGF4 deprivation of TSCs reduces geminin to a basal level that is required for maintaining endocycles in TGCs. Thus, geminin acts both like a component of the FGF4 signal transduction pathway that governs trophoblast proliferation and differentiation, and geminin is required to maintain endocycles. Geminin Is Essential to Prevent DNA Re-Replication-Dependent Apoptosis in Pluripotent Cells, but not in Differentiated Cells. Geminin is a dual-function protein unique to multicellular animals with roles in modulating gene expression and preventing DNA re-replication. We showed that geminin is essential at the beginning of mammalian development to prevent DNA re-replication in pluripotent cells, exemplified by embryonic stem cells, as they undergo self-renewal and differentiation. Depletion of geminin under conditions that promote either self-renewal or differentiation rapidly induced DNA re-replication, followed by DNA damage, then a DNA damage response, and finally apoptosis. Once differentiation had occurred, geminin was no longer essential for viability, although it continued to contribute to preventing DNA re-replication induced DNA damage. No relationship was detected between expression of geminin and genes associated with either pluripotency or differentiation. Thus, the primary role of geminin at the beginning of mammalian development is to prevent DNA re-replication-dependent apoptosis, a role previously believed essential only in cancer cells. These results suggest that regulation of gene expression by geminin occurs only after pluripotent cells differentiate into cells in which geminin is not essential for viability. Cytoplasmic localization of p21 protects trophoblast giant cells from DNA damage induced apoptosis. Proliferating trophoblast stem cells (TSCs) can differentiate into nonproliferating but viable trophoblast giant cells (TGCs) that are resistant to DNA damage induced apoptosis. We showed that p27 also remains localized to the nucleus during TSC differentiation where it complements the role of p57. Unexpectedly, p21 localized to the cytoplasm where it prevented DNA damage induced apoptosis. This unusual status for a Cip/Kip protein was dependent on site-specific phosphorylation of p21 by the Akt1 kinase that is also up-regulated in TGCs. Although cytoplasmic p21 is widespread among cancer cells, among normal cells it has been observed only in monocytes. The fact that it also occurs in TGCs reveals that p57 and p21 serve nonredundant functions, and suggests that the role of p21 in suppressing apoptosis is restricted to terminally differentiated cells.